The antibiotic thiolactomycin inhibits the growth of Escherichia coli K-12 and Pseudomonas aeruginosa 507 (β-lactam supersensitive mutant). A micrograph of E. coli cells, which were grown at a sublethal concentration of thiolactomycin (20μg/ml), revealed the morphological change with cell elongation. The effects of the antibiotic on syntheses of cellular constituents were studied by measuring the incorporation of labeled precursors into lipids and macromolecules. This antibiotic preferentially inhibited the incorporation. of [14C] acetate into fatty acids and lipids. Addition of both palmitate and oleate, but not of either fatty acid alone, reversed the growth inhibition of P. aeruginosa by thiolactomycin. These findings support the conclusion that the effects of thiolactomycin are due to a specific inhibition of fatty acid synthetase.